1. Field of the Invention
The invention relates generally to metalloprotease regulation of myostatin activity, and more specifically to methods of using agonists or antagonists of the BMP-1/TLD family of metalloproteases to modulate myostatin activity including, for example, to regulate muscle development in an organism, to methods of identifying agonists and antagonists of such metalloproteases, and to agonists and antagonists so identified.
2. Background Information
Myostatin is a transforming growth factor-β (TGF-β) family member that is essential for proper regulation of skeletal muscle growth. Myostatin is a secreted protein that is expressed specifically by cells of the skeletal muscle lineage during embryonic development and in adult animals; low levels of myostatin mRNA also are present in fat cells in adults animals. During early embryogenesis, myostatin mRNA is detectable in the myotome compartment of developing somites. At later embryonic stages and in postnatal life, myostatin is expressed widely in all skeletal muscles that have been examined.
The function of myostatin was elucidated by gene targeting studies in mice. Mice lacking myostatin demonstrated a dramatic and widespread increase in skeletal muscle mass due to muscle fiber hyperplasia and hypertrophy, indicating that myostatin is a negative regulator of muscle growth. The myostatin gene is highly conserved across evolution, with the predicted mature myostatin protein sequence being identical among mice, rats, humans, chickens, turkeys, and pigs, and highly homologous even with respect to aquatic organisms. The function of myostatin also is conserved, with mutations in the myostatin gene correlating to the double muscling phenotype in cattle.
The role of myostatin in regulating muscle growth and development indicates that methods and compositions that regulate myostatin activity can have a broad variety of applications, including, for example, for treating human diseases and for improving livestock production. With respect to human therapeutic applications, inhibitors of myostatin expression or function can provide a clinical benefit in the treatment of muscle wasting disorders such as muscular dystrophy, cachexia, and sarcopenia. In addition, myostatin deficient animals have a significant reduction in fat accumulation, and the loss of myostatin is protective against the development of obesity and type II diabetes in genetic models in mice. As such, modulation of myostatin activity also can be useful in the treatment of metabolic disorders such as obesity and type II diabetes. Further in this respect, inhibitors of myostatin expression or function not only can be useful for increasing the efficiency of livestock production, but also can result in the production of meat with a lower fat content.
Various strategies for manipulating the biological activities of myostatin have been described. Myostatin is synthesized as a precursor protein that undergoes proteolytic processing to generate an N-terminal fragment termed the “pro peptide” and a C-terminal fragment, a disulfide-linked dimer of which is the biologically active species. Currently described strategies for inhibiting myostatin activity have utilized molecules that can bind the myostatin C-terminal dimer and inhibit its activity. For example, myostatin binds two activin type II receptors, Act RIIA and Act RIIB, in vitro, and expression of a truncated dominant negative form of Act RIIB in transgenic mice resulted in the mice having increases in muscle mass comparable to that of transgenic myostatin knock out mice.
The myostatin pro peptide also has been used to inhibit myostatin activity. Following proteolytic processing, the myostatin pro peptide remains non-covalently associated with the C-terminal dimer and maintains the dimer in a latent, inactive state. The pro peptide has been shown to block the activity of the purified myostatin C-terminal dimer in various in vitro assays, and overexpression of the pro peptide in transgenic mice resulted in a phenotype characteristic of the myostatin null mutation. Follistatin is another protein that acts as a myostatin inhibitor. Follistatin can bind and inhibit the activity of a variety of TGF-β family members, including myostatin, and transgenic mice overexpressing follistatin in muscle have dramatic increases in muscle growth, consistent with inhibition of myostatin activity.
The above described inhibitors of myostatin each specifically interact with mature myostatin to inhibit its activity. While inhibiting the activity of a protein such as myostatin using an agent that directly interacts with the protein provides great specificity, such a method can require that all or most of the proteins be bound by the agent for the inhibitory effect to be manifest. An alternative way to inhibit the activity of a protein, particularly a protein that, itself must be activated by a second protein such as an enzyme in order for the first protein to be functional, is to target the second protein. Such a method can be advantageous because activating proteins such as enzymes generally are present at much lower levels than their substrates. As such, there is a greater likelihood that all or most of an activating protein such as an enzyme can be inhibited.
With respect to myostatin, at least two proteases are known to be involved in processing promyostatin, the primary gene product, into a signal peptide, a pro peptide and a C-terminal fragment, the latter of which forms homodimers that have biological myostatin activity. Unfortunately, these proteases also can act on a variety of other proteins and, therefore, agents that target and inhibit these proteases, for example, signal peptidase, likely would have diverse and deleterious effects if administered to a living organism. Thus, a need exists to identify biological molecules that are more specifically involved in regulating myostatin activation and activity. The present invention satisfies this need and provides additional advantages.